Immunoassays provide a means for determining whether an individual has been exposed to a pharmacological agent, a pathogen, or a substance of abuse (such as cocaine, opiates, etc.). Such determinations are of broad importance in medicine and law enforcement.
Immunoassays, are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular target molecule. Antibodies are immunoglobulins that are produced in response to the detection of a foreign substance ("antigen") within an animal. The region of an antigen that is recognized by an antibody, and which to which the antibody binds is referred to as an "epitope." Although large molecules, such as proteins or other molecules possess multiple epitopes, low molecular weight molecules, such as most pharmacological agents possess only a single epitope. Such low molecular weight molecules are referred to as "haptens." Immunoassays are used extensively in modern diagnostics (Fackrell, J. Clin. Immunoassay 8:213-219 (1985)). A large number of different immunoassay formats have been described (Yolken, R. H., Rev. Infect. Dis. 4:35 (1982); Collins, W. P., In: Alternative Immunoassays, John Wiley & Sons, New York (1985); Ngo, T. T. et al., In: Enzyme Mediated Immunoassay, Plenum Press, New York (1985)). Immunoassay formats have been developed that are amenable for large scale usage (for review, see Lee, T. T. T. et al., European Patent Application Publn. No. 203,238, herein incorporated by reference).
The simplest immunoassay involves merely incubating an antibody that is capable of binding to a predetermined molecule (i.e. an "analyte") with a sample that is suspected to contain the analyte. The presence of the target molecule is determined by the presence, and is proportional to the concentration, of any immune complexes that form through the binding of antibody and analyte. In order to facilitate the separation of such immune complexes from the unbound antibody initially present, a solid phase is typically employed. In more sophisticated immunoassays, the concentration of the target molecule is determined by binding the antibody to a support, and then incubating the bound antibody in the presence of the analyte-containing sample.
Target molecules that have become bound to the immobilized antibody can be detected in a variety of ways. For example, the support can be incubated in the presence of a labeled, second antibody (i.e. a "sandwich" immunoassay) that is capable of binding to a second epitope of the target molecule. Immobilization of the labeled antibody on the support thus requires the presence of the target, and is proportional to the concentration of the target in the sample. In an alternative assay, the sample is incubated with a known amount of labeled target and antibody binding site. Any target molecules present in the sample compete with the labeled target molecules for the antibody binding sites. Thus, the amount of labeled target molecules that are able to bind the antibody is inversely proportional to the concentration of target molecule in the sample. This is known as a competitive immunoassay.
The various immunoassay formats can be further divided into two main classes, depending upon whether the assay requires the separation of bound species from unbound species. Heterogeneous immunoassays require such purification, and hence entail a separation or isolation step. In contrast, homogeneous assays are designed such that the removal of bound from unbound species is unnecessary. Because homogeneous assays lack a separation step, and are more easily automated, they are more desirable than heterogeneous assays in applications that entail the screening of large numbers of patients.
One factor that attenuates the sensitivity of immunoassays is the degree of inaccessibility of the analyte being assayed. Analytes such as membrane associated proteins or liposaccharides are often significantly inaccessible to antibodies, and hence, their presence in a biological sample may escape detection. One partial solution to this problem has been to extract such insolubilized molecules using detergents. For example, Bowden, R. A. et al. discuss a turbidometric latex inhibition assay for detergent-solubilized lipopolysaccharides as a means for evaluating Brucella vaccine preparations (Bowden, R. A. et al., J. Microbiol. Meth. 16:297-306 (1992)). The use of detergents in enzyme linked immunosorbent assays is reviewed by Goldring, O. L. (Immunoassay Technol. 2:189-214 (1986)). Detergents have also been used in immunoassays of human immunodeficiency virus (HIV) and hepatitis B virus in order to inactivate the virus, and render the assay less hazardous to health care professionals (Bibb, W. et al., Abstr. Gen. Meet. Amer Soc. Microbiol. 91:396 (1991)). Detergents have also been used to facilitate the immobilization of antigens in agglutination immunoassays (Dorsett, P. H., U.S. Pat. No. 4,695,537).
Unfortunately, although the presence of detergent increases analyte accessibility, it decreases the rate and extent of immune complex formation (Goldring, O. L., Immunoassay Technol. 2:189-214 (1986); McCabe, J. P. et al., J. Immunol. Meth. 108:129-135 (1988); Noorduyn, L. A., J. Immunoassay 10:429-448 (1989)). Thus, the use of detergents has significant adverse consequences.
Regardless of immunoassay format, the utility of an immunoassay in detecting an analyte depends upon its capacity to report the extent of the formation of immune complexes between the antibody employed and the analyte whose presence or concentration is being measured. In general, two independent approaches exist for increasing this capacity. The first approach involves labeling one or more of the reagents. The second approach involves increasing the size of the immune complex.
A wide array of labels (such as radioisotopes, enzymes, fluorescent moieties, chemiluminescent moieties, or macroscopic labels, such as beads, etc.) have been employed in order to facilitate the detection of immune complexes (see, Chard., T., et al., In: Laboratory Techniques and Biochemistry in Molecular Biology (Work, T. S., Ed.), North Holland Publishing Company, New York (1978); Kemeny, D. M. et al. (Eds.), ELISA and Other Solid Phase Immunoassays, John Wiley & Sons, New York (1988)). Radioisotopes have long been used in immunoassays. O'Leary, T. D. et al., for example describe a radioimmunoassay ("RIA") for digoxin serum concentrations (O'Leary, T. D. et al., Clin. Chem. 25:332-334 (1979)). RIAs have the advantages of simplicity, sensitivity, and ease of use. Radioactive labels are of relatively small atomic dimension, and do not normally affect reaction kinetics. Such assays suffer, however, from the disadvantages that, due to radioisotopic decay, the reagents have a short shelf-life, require special handling and disposal, and entail the use of complex and expensive analytical equipment. The difficulty of handling such hazardous materials, and the problem of radioactive decay have led to the development of immunoassays that use other labels.
Enzymes, in particular, are now widely used as labels in immunoassay formats. Enzyme-linked immunoassays ("ELISAs") have the advantage that they can be conducted using inexpensive equipment, and with a myriad of different enzymes, such that a large number of detection strategies--colorimetric, pH, gas evolution, etc.--can be used to quantitate the assay. In addition, the enzyme reagents have relatively long shelf-lives, and lack the risk of radiation contamination that attends to RIA use. ELISAs are described in ELISA and Other Solid Phase Immunoassays (Kemeny, D. M. et al., Eds.), John Wiley & Sons, New York (1988), incorporated by reference herein. An enzyme-multiplied immunoassay technique (EMIT.RTM., Syva Co.) has been used to analyze the presence of analytes in biological fluids. The procedure is based on a competition between an analyte and an analyte-enzyme conjugate, for binding sites on an antibody present in limiting amounts (Cone, E. J. et al., J. Forens. Sci. 35:786-781 (1990); Baugh, L. D. et al., J. Forens. Sci. 36:79-85 (1991); Standefer, J. C. et al., Clin. Chem. 37:733-738 (1991); Schwartz, J. G. et al., Amer. J. Emerg. Med. 9:166-170 (1991);Helper, B. et al., Amer. J. Clin. Pathol. 81:602-610 (1984); Cambell, R. S. et al., J. Clin. Chem. Clin. Biochem. 24:155-159 (1986); Khanna, P., U.S. Pat. No. 5,103,021).
In addition to enzymes, fluorescent moieties are frequently used as labels. A fluorescence polarization immunoassay format (TDx.RTM., Abbott Laboratories, Inc.) has been found to be approximately equivalent to the EMIT.RTM.formats (Schwartz, J. G. et al., Amer. J. Emerg, Med. 9:166-170 (1991); Koizumi, F. et al., Tohoku J. Exper. Med. 155:159-(1988); Edinboro, L. E. et al., Clin. Toxicol. 29:241-(1991); Okurodudu, A. O. et al., Clin. Chem. 38:1040 (1992); Okurodudu, A. O. et al., Clin. Chem. 38:1040 (1992); Klotz, U., Ther. Drug. Monitor. 15:462-464 (1993)). Wong, S. H. Y., et al., have described the use of an automated (OPUS.TM.) analyzer to measure digoxin concentration in a monoclonal antibody mediated, fluorescence-based assay protocol (Wong, S. H. Y. et al., Clin. Chem. 38:996 (1992)). Lee, D. H. et al. also disclose the use of a fluorescence polarization assay and a chemiluminescent assay format to assay digoxin levels (Lee, D. H. et al., Clin. Chem. 36:1121 (1990)).
As indicated, immunoassay sensitivity can be enhanced by increasing the size of the immune complex that is formed in the immunoassay. If the immune complex is large enough, it will become capable of scattering light, or of spontaneously precipitating. In such cases, agglutination, or nephelometric or turbidimetric immunoassay methods may be employed. Nephelometric methods measure the light scattered by a suspension of particles or reflected toward a detector that is not in the direct path of light (Sternberg, J. C., Clin. Chem. 23:1456-1464 (1977)). In contrast, turbidimetric methods measure the reduction of light transmitted through the suspension of particles or aggregates. The reduction is caused by reflection, scatter, and absorption of the light by the aggregates. In both nephelometry and turbidimetry, the rate of change in light scatter may also be measured, and provides an indication of the amount of antigen present. Agglutination assays measure the precipitation of antibody-antigen complexes. Such assays can be extremely sensitive, and are amenable to automation. Because nephelometric and turbidimetric methods do not require the separation of the initially present antibody from the immune complexes formed in the assay, such assays are homogenous immunoassays. An agglutination inhibition assay for cocaine is commercially available (OnTrak.TM., Hoffman-LaRoche) but appears to be substantially less efficient than the above methods (Schwartz, J. G. et al., Amer. J. Emerg. Med. 9:166-170 (1991)).
The requirement of producing large immune complexes has limited the applicability of nephelometric, turbidometric or agglutination immunoassays to high molecular weight molecules, such as proteins, that possess several epitopes. In particular, since many pharmacological agents have only a single epitope, they are incapable of forming the large immune complexes needed for such immunoassays.
One approach to this problem involves the agglutination of antibody-coated particles with a polyepitopic species (or a developer antigen) containing at least two covalently coupled hapten analogs (e.g., a protein carder, such as bovine serum albumin ("BSA") (Mongkolsirichaikul, D. et al., J. Immunol. Meth: 157:189-195 (1993)). The agglutination reaction requires the use of a polyepitopic species or a developer antigen because a molecule that has only one epitopic site cannot bind two antibodies, and hence cannot cross-link two antibodies together. Such cross-linking is an essential step in the formation of large immune complexes. A second approach involves the agglutination of hapten-coated particles.
In either method, the hapten or drug in the sample competitively binds to the antibody binding sites and results in inhibition or reduction of the immunoagglutination. Particle agglutination assays for therapeutic drugs and drugs of abuse which use hapten coated particles are commercially available. Examples of such assays are PETINIA.TM. (Du Pont) and AbuScreen.TM. (Roche), Advisor.TM. (Abbott) and that of Mitsubishi.
A solution to this problem has recently been described by Oh, C. S. et al. in U.S. Pat. No. 5,168,057, by Harris, P. C. et al. in U.S. Pat. No. 5,196,351, and by Oh, C. S. et al., In: Nonisotopic Immunoassay, Ngo, T. T. (Ed.), Plenum Press, New York, pp. 457-476 (1988), all herein incorporated by reference, and involves the use of bidentate or tridentate analyte reagents. In bidentate immunoassay methods, the immune complex forms through two distinct binding reactions. One reaction involves the binding of the biotin member of the bidentate to an anti-biotin antibody (or a fragment of such an antibody), or to streptavidin or avidin. The other reaction is the immunoreaction of an anti-analyte antibody to the analyte member of the bidentate. Because antibody has two hapten binding sites, and avidin has four biotin binding sites, an immunocomplex is formed when the antibody, bidentate reagent, and avidin are mixed together. The formation of the immunocomplex is rapid and appears to be associated with the positive charge (pI 10) of the approximately thirty five lysine termini of avidin in addition to the strong binding of avidin and biotin. This specific charge-assisted immunoprecipitin reaction is a characteristic feature of the biotin-avidin methodology. Under similar conditions, streptavidin (pI 5) or charge neutralized avidin fails to produce the immunoprecipitin reaction with high rate.
In practice, steric hindrance between the F(ab) portion of the antibody (i.e., the hapten binding portion of the antibody) and avidin may block some of the avidin's four biotin-binding sites. Such blockage restricts both the rate and extent of immune complex formation. For example, if the spacer length between the hapten and biotin is less than about 27 .ANG., steric hindrance between avidin and the Fab portion of the antibody will block two of avidin's four biotin binding sites (Oh, C. S. et al., In: Nonisotopic Immunoassay, Ngo, T. T. (Ed.), Plenum Press, New York, pp. 457-476 (1988)). Such blockage causes the immune complex to be linear. The linear polymer results in a turbidity change or in light scattering which can be monitored on a turbidimeter or nephelometer, respectively. If hapten is present in the sample, it will compete for antibody with the hapten member of the bidentate reagent. Such competition leads to a reduction in the rate of immune complex formation. Thus, the rate of the nephelometric or turbidimetric response becomes inversely proportional to the concentration of the hapten in the sample.
Despite the success of the methods of Oh, C. S. et al. (U.S. Pat. No. 5,168,057) and Harris, P. C. et al. (U.S. Pat. No. 5,196,351) methods that would further improve both the rate of immune complex formation, and the resultant dose response of the immunoassay would provide more efficient and effective immunoassays for determining the concentration of medically important pharmacological agents. The present invention provides reagents and methods for conducting such improved immunoassays.